This invention relates to diagnosis and treatment of neoplastic diseases. More specifically, this invention provides methods useful for identifying agents which affect the complex signaling events involved in motility, morphological and potentially neoplastic cellular changes, including apoptotic cell death.
Various scientific and scholarly articles are cited throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
Cellular transformation during the development of cancer involves multiple alterations in the normal pattern of cell growth regulation. Primary events in the process of carcinogenesis involve the activation of oncogene function by some means (e.g., amplification, mutation, chromosomal rearrangement), and in many cases, the removal of anti-oncogene function. In the most malignant and untreatable tumors, normal restraints on cell growth are completely lost as transformed cells escape from their primary sites and metastasize to other locations in the body. One reason for the enhanced growth and invasive properties of some tumors may be the acquisition of increasing numbers of mutations in oncogenes, with cumulative effect (Bear et al., Proc. Natl. Acad. Sci. USA 86:7495-7499, (1989)). Alternatively, insofar as oncogenes function through the normal cellular signaling pathways required for organismal growth and cellular function (reviewed in McCormick, Nature 363:15-16, (1993)), additional events corresponding to mutations or deregulation in the oncogenic signaling pathways may also contribute to tumor malignancy (Gilks et al., Mol. Cell Biol. 13:1759-1768, (1993)), even though mutations in the signaling pathways alone may not cause cancer.
Several discrete classes of proteins are known to be involved in bringing about the different types of changes in cell division properties and morphology associated with transformation. These changes can be summarized as, first, the promotion of continuous cell cycling (immortalization); second, the loss of responsiveness to growth inhibitory signals and cell apoptotic signals; and third, the morphological restructuring of cells to enhance invasive properties.
Of these varied mechanisms of oncogene action, the role of control of cell morphology is one of the least understood. Research using non-transformed mammalian cells in culture has demonstrated that simply altering the shape of a cell can profoundly alter its pattern of response to growth signals (DiPersio et al., Mol. Cell Biol. 11:4405-4414, (1991)), implying that control of cell shape may actually be causative of, rather than correlative to, cell transformation. For example, mutation of the antioncogene NF2 leads to development of nervous system tumors. Higher eucaryotic proteins involved in promoting aberrant morphological changes related to cancer may mediate additional functions in normal cells that are not obviously related to the role they play in cancer progression, complicating their identification and characterization.
Recent evidence suggests that certain key proteins involved in control of cellular morphology contain conserved domains referred to as SH2 and SH3 domains. These domains consist of non-catalytic stretches of approximately 50 amino acids (SH3) and 100 amino acids (SH2, also known as the xe2x80x9cSrc homology domainxe2x80x9d). SH2/SH3 domains are found in cytoskeletal components, such as actin, and are also found in signaling proteins such as Abl. The interaction of these proteins plays a critical role in organizing cytoskeleton-membrane attachments.
Besides the numerous SH2/SH3-containing molecules with known catalytic or functional domains, there are several signaling molecules, called xe2x80x9cadapter proteins,xe2x80x9d which are so small that no conserved domains seem to exist except SH2 and SH3 domains. Oncoproteins such as Nck, Grb2/Ash/SEM5 and Crk are representatives of this family. The SH2 regions of these oncoproteins bind specific phosphotyrosine-containing proteins by recognizing a phosphotyrosine in the context of several adjacent amino acids. Following recognition and binding, specific signals are transduced in a phosphorylation dependent manner.
As another example, P47v-Crk (CrK) is a transforming gene from avian sarcoma virus isolate CT10. This protein contains one SH2 and one SH3 domain, and induces an elevation of tyrosine phosphorylation on a variety of downstream targets. One of these targets, p130cas, is tightly associated with v-Crk. The SH2 domain of v-Crk is required for this association and subsequent cellular transformation. P130cas is also a substrate for Src mediated phosphorylation. Judging from its structure, p130cas may function as a xe2x80x9csignal assemblerxe2x80x9d of Src family kinases and several cellular SH2-containing proteins. These proteins bind to the SH2 binding domain of p130cas, which is believed to induce a conformational change leading to the activation and inactivation of downstream signals, modulated by multiple domains of the protein.
Another oncogene, Ras, is a member of a large evolutionarily conserved superfamily of small GTP-binding proteins responsible for coordinating specific growth factor signals with specific changes in cell shape, including the development of stress fibers and membrane ruffles (Ridley and Hall, Cell 70:389-399, 1992; Ridley et al., Cell 70:401-410,(1992)). A rapidly growing family of oncoproteins, including Vav, Bcr, Ect-2, and Dbl, has been found to be involved in a variety of different tumors (Eva and Aaronson, Nature 316:273-275, (1985); Ron et al., EMBO J. 7:2465-2473, (1988); Adams et al., Oncogene 7:611-618, (1992); Miki et al., Nature 362:462-465, (1993)). Proteins of this family have been shown to interact with Ras/Rac/Rho family members, and possess sequence characteristics that suggest they too directly associate with and modulate organization of the cytoskeleton.
In view of the significant relationship between signaling or xe2x80x9cadapterxe2x80x9d proteins, altered cellular morphology and the development of cancer, it would be of clear benefit to identify and isolate such proteins (or genes encoding them) for the purpose of developing diagnostic/therapeutic agents for the treatment of cancer.
It is an object of the present invention to provide a purified nucleic acid molecule of human origin that encodes a signal mediator protein (SMP) also known as HEF-1, involved in the signaling cascade related to morphological cellular changes, and therefrom provide isolated and purified protein for use in methods for identifying beneficial therapeutic agents which regulate these signaling cascades. Such methods facilitate the identification and characterization of other genes and proteins involved in regulating cellular morphology, division and death.
HEF-1 is encoded by SEQ ID NO: 1 and is described and claimed in U.S. Pat. No. 5,716,782. The amino acid sequence of HEF-1 is provided in SEQ ID NO:2 and is described and claimed in U.S. application Ser. No. 09/196,466. Antibodies immunologically specific for HEF-1 are disclosed in U.S. Pat. No. 6,100,384. All of the foregoing patents and patent applications are incorporated by reference herein in their entirety.
In accordance with the present invention, HEF-1 has been implicated in a variety of fundamental biological processes. These include, apoptosis, T cell and epithelial cell migration, and cell spreading. Various assays are provided herein for identifying therapeutic agents which regulate these processes.
Thus, in one embodiment of the invention, methods are provided for identifying test compounds which regulate apoptosis mediated by HEF1. An exemplary method involves incubating HEF1 overexpressing cells in the presence of a test compound suspected of having apoptotic regulatory activity relative to untreated controls and assessing whether the test compound alters the number of cells undergoing programmed cell death. Cell death may be assessed by a variety of methods, including without limitation, trypan blue assay, MTT assay, propridium iodide uptake, DNA fragmentation or laddering, alterations in cellular morphology and the generation of caspase cleavage products.
In another embodiment of the invention, methods are provided for identifying test compounds which affect cellular motility. An exemplary method involves incubating HEF1 overexpressing cells in the presence of a test compound suspected of having motility regulating activity relative to untreated controls and assessing whether the test compound impedes or promotes cellular motility. Cellular motility or migration may be assessed using a variety of techniques which include without limitation, Boyden two chamber assays and quantitation using Isee(copyright) imaging software.
In a further embodiment of the invention, methods are provided for determining whether germline or somatic cell samples from patients contain altered HEF1 encoding nucleic acids. Such methods include without limitation, SSCP, direct sequencing, and heteroduplex formation followed by cleavage by mismatch recognizing enzymes followed by electrophoresis.
In a final embodiment, kits for performing the foregoing methods are also provided.